刺参甘露糖结合凝集素的生物信息学分析

甘露糖结合凝集素(mannan-binding lectin,MBL)属于C型动物凝集素超家族,特异地结合甘露糖。MBL是宿主起始免疫系统一个重要成分,因此近年来逐渐成为研究的热点。采用生物信息学技术对刺参(Apostichopus japonicus)MBL(AJ-MBL)的两个基因编码的蛋白质结构和功能进行了分析,包括信号肽、分子量、等电点、跨膜结构域、糖基化和磷酸化位点、二级结构和三级结构等,旨在了解其结构特征,为动物免疫反应方面研究提供理论基础。     

甘露糖结合凝集素基因多态性与结核病的相关性研究进展

结核病的发生是结核分枝杆菌与宿主之问相互作用的结果.并受环境因素影响.人们时结核病的研究重点逐渐转移到其宿主的易感基因上来,其中甘露糖结核凝集素(MBL)基因是当今国内外研究的热点之一.MBL通过结合病原生物表面的甘露糖等糖基受体而直接介导调理吞噬作用和/或通过MBL途径激活补体,在机体的固有性免疫防御中发挥重要作用.本文主要介绍MBL基因多态性与结核病易感性的研究进展.     

MBL与Raji细胞结合特性的研究

甘露聚糖结合凝集素(MBL)作为关键的天然免疫模式识别分子已得到共识,但其在获得性免疫应答中是否发挥作用目前尚不清楚.采用酶联免疫吸附试验分析MBL能否与Raii、THPI/CDl4、Jurkat和红细胞结合,并采用流式细胞术着重研究其与Raji细胞结合特性.结果显示:MBL以浓度依赖方式结合Raji、THP1/CDl4、Jurkat细胞,红细胞则否.MBL与Raji细胞结合是Ca2 依赖的,且能被甘露糖、葡萄糖、N-乙酰葡糖胺所抑制;Clq或抗ClqR单克隆抗体能部分抑制MBL与Raji细胞结合;重组人MBL-CRD蛋白或MBL-CLR蛋白均能抑制MBL与Raji细胞结合,两者联合应用则可完全阻断这种结合.研究资料表明,B淋巴细胞系Raji细胞表达Ca2 依赖性、糖敏感的MBL受体,包括对CLR特异和CRD特异的两种受体,前者为MBL和Clq的共同受体.进一步的功能研究显示,高浓度MBL(10-50mg/L)对Raji细胞的生长具有显著抑制作用,且呈剂量依赖关系.提示MBL作为一种重要的模式识别分子,不仅发挥天然免疫功能,而且可能在调节获得性免疫应答中起一定作用.     

红花菜豆（矮生红花变种）凝集素的生物学作用

红花菜豆（矮生红花变种,Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒ．ｒｕｂｒｏｎａｎｕｓ）凝集素（ＰＣＬ）识别高甘露糖型糖并与之结合,它的生物学作用都与其糖结合专一性有关,ＰＣＬ的血凝作用不显示供血动物专一性和和血型专一性,ＰＣＬ除凝集红细胞外亦凝集动物其它细胞,如小鼠脾细胞,人精细胞及某些肿瘤细胞如黑色素瘤细胞Ｍ２１,胃癌细胞等,此外ＰＣＬ亦凝集某些微生物如野生型和Ｈ．Ｂ．１０１大肠杆菌以及面包     

半夏凝集素的糖结合活性研究

半夏凝集素可与甘露聚糖结合。本文以ＰＴＬ与＾１２５Ｉ标记的甘露聚糖的结合活性为指标,观察了一些金属离子对ＰＴＬ的糖结合活性的影响,并对ＰＴＬ的糖结合专一性作了较系统的研究。结果表明常见的金属离子或ＥＤＴＡ对其糖结合活性无显著影响,但Ｋ＾＋可明显增加ＰＴＬ的糖结合活性。大多数单糖,二糖不抑制ＰＴＬ与甘露聚糖的结合,但一些疏水配基形成的糖苷可产生显著的抑制效应。ＰＴＬ专一与高甘露糖型糖链结合。     

凝集素在食源致病菌快速检测中应用的研究进展

凝集素是一类具有独特糖专一性、能够与糖非共价可逆结合的蛋白质。食源致病菌表面存在大量糖蛋白分子,凝集素因能够与其发生高亲和力的结合,被广泛应用于食源致病菌的快速检测中。凝集素作为识别分子用于食源致病菌的分离与检测,能够改善检测新方法的实用性、消除食品基质的干扰、缩短样品的处理时间、提高检测的灵敏度。本文介绍了凝集素的基本信息及其糖特异性识别机制,并对凝集素在食源致病菌快速检测领域的应用进展进行了综述。     

半夏凝集素的糖结合活性研究

半夏凝集素(PTL)可与甘露聚糖结合。本文以PTL与~(125)I标记的甘露聚糖的结合活性为指标,观察了一些金属离子对PTL的糖结合活性的影响,井对PTL的糖结合专一性作了较系统的研究。结果表明常见的金属离子或EDTA对其糖结合活性无显著影响,但K~+可明显增加PTL的糖结合活性。大多数单糖,二糖不抑制PTL与甘露聚糖的结合,但一些疏水配基形成的糖苷可产生显著的抑制效应。PTL专一与高甘露糖型糖链结合。     

半夏蛋白的若干生物学性质

从半夏块茎鲜汁分离的半夏蛋白具有凝集细胞和促使细胞分裂的作用。在已测试的红细胞中凝集羊、狗、猫、兔、豚鼠、大鼠、小鼠和鸽的红细胞,但不凝集人、猴、猪和鸡、鸭、鹅、龟、蟾蜍、鳝的红细胞。半夏蛋白的血凝作用和已知的多种凝集素一样存在供血动物种属专一性。半抗原抑制试验结果,所测各种单糖、双糖、聚糖和糖蛋白中,只有甘露聚糖和含有寡聚甘露糖苷核心的甲状腺球蛋白有抑制作用,表明半夏蛋白只与甘露聚糖结合,且其分子上与糖互补的位置远远大于一个单糖分子的范围。是目前已知的唯一只与甘露糖而不与葡萄糖结合的一种具有凝集素作用的蛋白质。除红细胞外半夏蛋白亦凝集其它细胞,已测细胞中小鼠脾细胞、人肝癌细胞(QGY 7703-3和7402)、艾氏腹水癌和腹水型肝癌细胞均被半夏蛋白凝集,但它不凝集大鼠附睾和猪大网膜脂肪细胞,虽然能和这两种细胞结合。提示半夏蛋白的细胞凝集作用不仅具有动物种属专一性并存在细胞类别专一性。半夏蛋白的促细胞分裂作用亦有动物种属专一性,它促使兔外周血淋巴细胞转化,但不促使人外周血淋巴细胞分裂。     

半夏蛋白的若干生物学性质

从半夏块茎鲜汁分离的半夏蛋白具有凝集细胞和促使细胞分裂的作用。在已测试的红细胞中凝集羊、狗、猫、兔、豚鼠、大鼠、小鼠和鸽的红细胞,但不凝集人、猴、猪和鸡、鸭、鹅、龟、蟾蜍、鳝的红细胞。半夏蛋白的血凝作用和已知的多种凝集素一样存在供血动物种属专一性。半抗原抑制试验结果,所测各种单糖、双糖、聚糖和糖蛋白中,只有甘露聚糖和含有寡聚甘露糖苷核心的甲状腺球蛋白有抑制作用,表明半夏蛋白只与甘露聚糖结合,且其分子上与糖互补的位置远远大于一个单糖分子的范围。是目前已知的唯一只与甘露糖而不与葡萄糖结合的一种具有凝集素作用的蛋白质。除红细胞外半夏蛋白亦凝集其它细胞,已测细胞中小鼠脾细胞、人肝癌细胞(QGY7703-3和7402)、艾氏腹水癌和腹水型肝癌细胞均被半夏蛋白凝集,但它不凝集大鼠附睾和猪大网膜脂肪细胞,虽然能和这两种细胞结合。提示半夏蛋白的细胞凝集作用不仅具有动物种属专一性并存在细胞类别专一性。半夏蛋白的促细胞分裂作用亦有动物种属专一性,它促使兔外周血淋巴细胞转化,但不促使人外周血淋巴细胞分裂。     

芝麻凝集素的纯化及其性质

凝集素作为研究细胞表面糖蛋白的结构和功能的探针,日益引起人们的兴趣。各种来源的凝集素可以有不同的糖结合专一性,这就为研究含糖高分子中糖部分的结构提供了一个有效的手段。我们在从各科植物种子中筛选凝集素时发现,芝麻抽提液含有能与多糖结合的蛋白     

Alpha-C-mannosyltryptophan is not recognized by conventional mannose-binding lectins

Alpha-C-mannosyltryptophan (C-Man-Trp) is a novel, naturally occurring C-linked carbohydrate-protein linkage first found in 1994 from human ribonuclease 2. Since then, a number of C-Man-Trp residue have been found from several important proteins such as interleukin 12 beta, components of complement system, thrombospondin-1, and erythropoietin receptor, however, the biological functions have remained unknown even though its biosynthetic pathway has been revealed. In order to find a clue as to the biological functions, we examined the affinity of C-Man-Trp with conventional mannose lectin such as concanavarin A (Con A) and mannose-binding lectin (MBL). The affinity of C-Man-Trp with Con A, a typical mannose-binding lectin from plant was examined using a Con A-Sepharose column. Unlike p-nitrophenyl-alpha-O-Man, C-Man-Trp was not retained on the column. MBL-C, a major mannose-binding lectin purified from mouse serum, did not bind with N-biotinylated C-Man-Trp, judging from ELISA based assay. These results imply that C-Man-Trp may be recognized with the other specific proteins associated with its unknown biological functions.     

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.     

Biological activities of human mannose-binding lectin bound to two different ligand sugar structures, Lewis A and Lewis B antigens and high-mannose type oligosaccharides.

The biological activities of mannose-binding lectin (MBL) which binds to different ligands on mammalian cells were examined using two types of Colo205 cells, a human colon adenocarcinoma cell line: one naturally expressing Lewis A and Lewis B antigens as ligands for MBL (NT-Colo205), and the other modified to express high-mannose type oligosaccharides by treatment with benzyl-2-acetamide-2-deoxy-alpha-galactopyranoside and 1-deoxymannojirimycin (Bz+dMM-Colo205). Although the final lysis was not observed, the deposition of C4 and C3 was observed on both types of Colo205 cells after treatment with MBL and complements as a result of complement activation by MBL. MBL bound to Bz+dMM-Colo205 could also activate human peripheral blood leukocytes and induce superoxide production; however, MBL bound to NT-Colo205 could not. This may be explained by the lower affinity of MBL to Lewis A and Lewis B antigens than to high-mannose type oligosaccharides under physiological conditions, since MBL bound to NT-Colo205 was more easily released from the cell surface than that bound to Bz+dMM-Colo205 at 37 degrees C. These findings suggest that the difference in the affinity of MBL to its ligands could influence the expression of some biological activities of MBL.     

Sugar-mediated ligand-receptor interactions in the immune system

Most molecules involved in the recognition and elimination of pathogens by the immune system are glycoproteins. Oligosaccharides attached to glycoproteins initiate biological functions through mechanisms that involve multiple interactions of the monosaccharide residues with receptors. For example, calreticulin, a quality-control lectin-like chaperone, interacts with glucosylated mannose glycans presented by empty major histocompatibility complex (MHC) class I molecules, retaining them in the endoplasmic reticulum (ER) until antigenic peptide is loaded. Clusters of specific IgG glycoforms, present in increased amounts in rheumatoid arthritis, bind mannose-binding lectin (MBL), providing a potential route to inflammation through activation of the complement pathway. Secretory IgA glycans bind gut bacteria, and an unusual cluster of mannose residues on gp120, the surface coat protein of the HIV virus, is recognized by the novel 'domain-swapped' IgG 2G12 serum antibody.     

Identification and characterization of a novel human collectin CL-K1

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.     

Disease-associated mutations in human mannose-binding lectin compromise oligomerization and activity of the final protein

Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement.     

Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages. A secondary advantage of these expression systems would be to eliminate the need for carbohydrate remodeling. Here, multiple expression systems were used to produce GCase containing primarily terminal oligomannose, from Man2 to Man9. GCase from these multiple expression systems was compared to Cerezyme with respect to affinity for mannose receptor and serum mannose-binding lectin (MBL), macrophage uptake, and intracellular half-life. In vivo studies comparing clearance and targeting of Cerezyme and the Man9 form of GCase were carried out in a Gaucher mouse model (D409V/null). Mannose receptor binding, macrophage uptake, and in vivo targeting were similar for all forms of GCase. Increased MBL binding was observed for all forms of GCase having larger mannose structures than those of Cerezyme, which could influence pharmacokinetic behavior. These studies demonstrate that although alternative cell expression systems are effective for producing oligomannose-terminated glucocerebrosidase, there is no biochemical or pharmacological advantage in producing GCase with an increased number of mannose residues. The display of alternative carbohydrate structures on GCase expressed in these systems also runs the risk of undesirable consequences, such as an increase in MBL binding or a possible increase in immunogenicity due to the presentation of non-mammalian glycans.     

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus,Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.     

Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response against bacterial lipopolysaccharide (LPS) stimulation. LPS-mediated pro-inflammatory cytokine productions on mBMMCs obtained from Toll-like receptor4 (TLR4)-deficient mice, TLR2-defficient mice, and their wildtype, were specifically attenuated by the addition of either mouse MBL-A or ficolin-A in a dose-dependent manner. However, the inhibitory effects by mouse MBL-A or ficolin-A were restored by the addition of mannose or N-acetylglucosamine, respectively. These results suggest that mouse MBL-A and ficolin-A bind to LPS via its carbohydrate-recognition domain and fibrinogen-like domain, respectively, whereby cytokine production by LPSmediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells. [BMB Reports 2013; 46(7): 376-381]